This invention relates to blood fractionation and more particularly to the production of blood coagulation Factor VIII:C.
The process of blood coagulation is a complicated physiological activity that involves interactions of numerous substances found in normal whole blood. It is known that certain factors associated with the blood coagulation mechanism are seriously deficient in certain individuals. Thus, in those patients suffering from classical hemophilia, antihemophilic factor A (AHF, Factor VIII) is deficient. In those patients suffering from hemophilia B, plasma thromboplastin component (PTC, Factor IX) is missing from the blood. A small percentage of hemophiliacs also are lacking in the so-called Von Willebrand Factor which is an integral component of Factor VIII.
It is now generally recognized that plasma Factor VIII is a complex of two components that have distinct functions, biochemical and immunological properties, and genetic control. One component of the Factor VIII complex has antihemophilic factor procoagulant activity and is usually designated Factor VIII:C. The other, larger component comprises the majority of the protein mass, interacts with platelets in a way that promotes primary hemostasis and is usually designated Factor VIIIR (ristocetin cofactor or Von Willebrand antigen).
Patients with Factor VIII:C deficiency transmitted by X-chromosome inheritance (hemophilia A patients) have normal Factor VIIIR synthesis and function. Such patients thus do not require exogenous administration of Factor VIIIR for maintenance of hemostasis, and a concentrate of Factor VIII:C free of Factor VIIIR would be satisfactory and in some cases even preferable.
Further background information on the structure and function of the Factor VIII complex and its two components can be had by reference to the three recent review articles by, respectively, Hoyer, J. Amer. Soc. of Hematol. 58 (1), 1-13 (1981); Harris et al, Biochim. Biophys. Acta 668, 456-470 (1981); and Fulcher et al, Proc. Natl. Acad. Sci. USA 79, 1648-1652 (1982).
The clinical importance of Factor VIII concentrates and the critical need for adequate supplies thereof has provided motivation to develop improved methods for the production of such blood fractions. As alternatives to the conventional Cohn alcohol process of blood fractionation which must be conducted at cold temperatures, various other methods have been developed which employ fractionating agents that can be used at normal room temperature (ambient temperature). One such method employs the polymer polyethylene glycol (PEG) as described, e.g., in U.S. Pat. Nos. 3,631,018; 3,652,530; and 3,682,881. However, the methodology described in these patents additionally employs a cryoprecipitation step which necessitates the use of cold temperature facilitates and also results in a loss of a substantial amount of the Factor VIII activity.
Addition of heparin at various stages of these PEG/cryoprecipitation fractionation processes to increase the yield of Factor VIII has been suggested in U.S. Pat. Nos. 3,803,115; Re. 29,698; 4,203,891; and 4,289,691. In the first two of these patents the heparin is added after the cryoprecipitation step whereas in the latter two patents it is added before the cryoprecipitation step.
The aforesaid prior art methods for the production of Factor VIII by use of polyethylene glycol, cryoprecipitation and heparin are not reported to provide a Factor VIII:C concentrate as distinguished from the Factor VIII complex. However, heparin has been suggested for addition to plasma in fractionation methodology to separate AHF, von Willebrand's ristocetin cofactor and fibronectin by cold temperature precipitation and chromatography according to U.S. Pat. Nos. 4,210,580 and 4,278,594.
Another method of improvement over the conventional Cohn alcohol blood fractionation process employs water-insoluble, cross-linked polyelectrolyte copolymer adsorbents as described, e.g., in U.S. Pat. Nos. 3,554,985; 3,555,001; 4,118,554; and 4,157,431; and by A. J. Johnson, et al., J. Lab. Clin. Med. 92 (a), 194-210 (1978). These polymeric materials have been employed in combination with other agents such as dithiothreitol, Sepharose CL-4B and Sephadex G-100 to produce a concentrate of Factor VIII:C substantially free of Factor VIIIR. Harris et al., Biochim. Biophys. Acta 668, 456-470 (1981).